It has long been an objective in the field of photodynamic therapy to define the critical sites of photosensitizer uptake, binding and damage to permit the design and synthesis of more efficacious drugs. The development of a congeneric series of photosensitizers ( the pyropheophorbide ethers which have similar photophysical and photochemical properties but which localize to different subcellular sites and have a range of different photoactivities in vitro and in vivo), now provides an useful tool to probe the binding sites and determine which are most vulnerable to photodamage causing cell death. The overall goal of this Project is to identify a) the sites at the cellular level which enable optimal photosensitizer efficiency, b) the photosensitizing agents which interact with them and c) elucidate the mechanisms of photodamage occurring at those sites which may be important for future rational drug design. To acheive this goal, effective and less effective photosensitizers including the pyrophophorbide ethers and new congeneric series being developed in Project I will be examined for subcellular localization sites by fluorescent microscopy and for binding specificity by competition with specific ligands at sites thought to be important for photodynamic efficiency (particularly in mitochondria and at the peripheral benzodiazepine receptor). The physicochemical characteristics of the photosensitizers at the binding sites will be evaluated by spectrally resolved imaging and their capacity for causing lethal photodamage will be assessed by endpoints indicative of impaired functioning at mitochondrial sites (mitochondrial depolarization and triggering of the membrane permeability transition). In addition, agents capable of inhibiting and initiating physiological responses associated with the peripheral benzodiazepine receptor complex will be used to strengthen evidence of specific photodynamic reactions at that site by modulating the PDT response. Further, using histological staining techniques, correlative evidence for co-localization of photosensitizers and specific markers for sites sensitive to PDT will be sought in tissues from malignant and normal human archival and murine samples to support the theory that specific binding sites are present at higher levels in proliferating cells proving both selective and advantageous for effective PDT.